Method for identifying Listeria monocytogenes and culture medium

ABSTRACT

A substrate which can be used for the direct identification of pathogenic bacteria belonging to the genus  Listeria  by detecting an esterase activity other than Phosphatidyl Inositol-specific Phospholipase C (PI-PLC), an esterase which is specific to the species  Listeria monocytogenes.  The use of two substrates, one as described above and the other specific for all or some members of the genus  Listeria,  and a reaction medium containing such a substrate or such a combination of substrates are disclosed. An identification method which exploits such culture media is also disclosed. The invention is particularly applicable in the field of diagnosis.

This invention concerns a chromogenic substrate which can be used forthe direct identification of pathogenic bacteria belonging to the genusListeri, more precisely those of the species Listeria monocytogenes.

The invention also concerns using a combination of two substrates, onesubstantially specific for the species Listeria monocytogenes, and theother either specific or not for the genus Listeria. It also concerns areaction medium containing such a substrate or such a combination ofsubstrates. Finally, it concerns an identification method exploitingsuch culture media.

For many years, special substrates have been used to determine whethercertain enzyme activities typical of microorganisms are present or not.Through the use of specific substrates, it is possible—on the basis ofwhether a reaction takes place or not—to characterize the nature of agroup of microorganisms, or distinguish between different strains and/orspecies belonging to a given genus.

Synthetic enzyme substrates such as those exploited in this inventionare made up of two different parts: the first part is specific to theenzyme activity being tested for and will hereafter be referred to asthe target part; the second part acts as a marker and will hereafter bereferred to as the marker part.

Such special substrates may be either fluorescent or chromogenic. Infact, the second marker part or the product of its reaction with one ormore other compounds becomes fluorescent or chromogenic when it is nolonger bound to the first target part (in this context, refer to PatentApplication PCT/FR99/00781 filed on behalf of the applicant).

Isolating and identifying the bacterium Listeria monocytogenes is amajor problem in safety monitoring in both the food industry and medicalbacteriology. Among all the bacterial species belonging to the genusListeria, only Listeria monocytogenes is known to be pathogenic inhumans. It can cause listeriosis, a condition which is sometimes mortal(in 25 to 35% of cases) in the immunodeficient, very young children andpregnant women. Other Listeria species are non-pathogenic, or are onlypathogenic in animals. Such is the case in particular for Listeriaivanovii.

Although the danger of listeriosis has diminished in recent years inmost developed countries, modern society demands ever more stringentsafety and, even if sporadic cases are accepted, more widespreadoutbreaks are not.

However, in France, for example, risk management policy tends to focuson the products further down the line (e.g. contamination levels infinished products) rather than components higher up in the productionprocess (the infection of livestock). As a result, between 15 and 60% ofall poultry carcasses, between 3 and 36% of all pig carcasses, andbetween 7 and 28% of all cow carcasses are contaminated by Listeria.

Therefore, when it comes to human bacterial infection, it is importantto be able to make a definitive distinction between Listeriamonocytogenes and the other, non-pathogenic members of the genusListeria.

Distinction has traditionally been made between different Listeria spp.by using selective culture media. Two particular selective media are inthe most widespread use: Palcam (Van Netten et al., J. Food Microbiol.(1988), 6, pp. 187-188) and Oxford (Curtis et al., Lett. Appl.Microbiol. (1989), 8, pp. 85-98). All species belonging to the genusListeria can be detected using these media. Typical colonies areobtained which must subsequently be identified by means of furthertests—microscopic and/or biochemical and/or immunologic and/orgenetic—to check whether or not the colony corresponds to the speciesListeria monocytogenes.

These extra experiments both slow down and increase the cost of testing.Furthermore, they require many different reagents and necessitatetrained technicians. Finally, since picking the colonies to beidentified is a random process, the extra experiments often introducemistakes, or at least compromise the accuracy and reliability of theresult. This is a particularly acute problem when the number of coloniesof Listeria monocytogenes on the isolation medium is much lower than thenumber of colonies of other Listeria species.

The Applicant's Patent EP-B-0.496.680 describes a bacteriological testmethod to differentiate the species Listeria monocytogenes from otherbacterial species which belong to the genus Listeria. According to thismethod, use is made of an identification medium containing a chromogenicor fluorogenic substrate which is hydrolyzed by an enzyme called Glycineaminopeptidase. The medium used may also contain a substrate which canbe fermented and/or a substrate which can be reduced and/or a substratewhich can be enzymatically hydrolyzed, such as the substrate forα-mannosidase, the chemical conversion of which makes it possible tocharacterize the Listeria species present in the test sample.

This useful approach has one major drawback in that the species Listeriamonocytogenes is the only one not to express Glycine aminopeptidaseactivity. It is therefore possible to identify all Listeria speciesexcept the one member of the genus which is pathogenic, namely Listeriamonocytogenes. Trying to detect Listeria monocytogenes by virtue of theabsence of a certain activity is not very reliable since such a negativetest lacks specificity because of the possibility of mutation andbecause another species might not be expressing its normal pattern ofactivity due to stress.

Methods based on using chromogenic media to detect Listeriagenus-specific β-glucosidase activity have been developed. Moreover,other techniques based on chromogenic media can be used to distinguishbetween Listeria monocytogenes and Listeria ivanovii from other Listeriaspecies by assaying phosphatidylinositol phospholipase C (PI-PLC)activity.

It has been shown that certain species of the genus Listeria (such asListeria monocytogenes and Listeria ivanovii) secrete PI-PLC into theculture medium (Leimeister-Wächter et al., Mol. Microbiol. (1991) 5(2),pp. 361-366; J. Mengaud et al., Mol. Microbiol. (1991) 5(2), pp. 367-372; and Goldfine et al., Infection and Immunity (1992) 60(10), pp.4059-4067). It is also known that these two species can be identifiedusing indirect methods (Notermans et al., App. and Env. Microbiology(1991), vol. 57 n°9, pp. 2666-2670.

Patent Application WO-A-99/04032 describes a culture medium containing achromogenic substrate which is specific for Listeria monocytogenes andListeria ivanovii, in the form of a phosphatidylinositol derivative suchas the ammonium salt of5-Bromo-4-chloro-3-indolyl-myo-inositol-1-phosphate. This affords directdetection of both these species in a single step, and therefore makes itpossible to distinguish these two from all other Listeria species.

Bacteriological detection tests based on PI-PLC are also dealt with inthe following documents:

-   -   WO-A-98/38332 which focuses on a method and a detection test for        PI-PLC, based on the cleavage of a substrate by the enzyme, one        of the residues of said substrate being chromogenic and making        it possible to identify pathogenic Listeria, and    -   WO99/48899 which describes a fluorogenic substrate based on        4-methylumbelliferone which is used to detect the PI-PLC        activity expressed by a number of different species, namely        Clostridium spp., Listeria ivanovii, Staphylococcus aureus,        Bacillus cereus, Bacillus thuringiensis as well as Listeria        monocytogenes.

In this document, attention is drawn to the fact that phospholipase Cactivity is not specific for Listeria monocytogenes since it is alsofound in other species, including another Listeria species, namelyListeria ivanovii.

However, it should be noted that esterase substrates are more easily andmore cheaply synthesized than substrates for PI-PLC. Moreover, theadvantage of using a chromogenic substrate (rather than a natural one)is that visual detection is easier, i.e. it is easier to distinguishcolored colonies than it is to visualize a halo surrounding colonies.This advantage furthermore applies to cultures containing differentmicroorganisms in that each colony has its own specific color, whereas ahalo could spread out below two disparate colonies.

The purpose of this invention is to offer a detection method which makesit possible to differentiate the species Listeria monocytogenes from allother Listeria species. This is achieved by detecting at least onemetabolic activity which has not hitherto been used either to detectListeria or to differentiate Listeria monocytogenes from other speciesbelonging to the genus Listeria. This activity is an esterase which isexpressed far more strongly by Listeria monocytogenes than by otherspecies in the genus. In addition, at least one other activity isassayed, namely an activity which is expressed by all or some Listeriasuch as a saccharidase, a phosphatase or an aminopeptidase, whichenhances the contrast between the color of colonies of Listeriamonocytogenes and that of colonies of other species belonging to thesame genus. This makes it possible to distinguish Listeria monocytogenesfrom Listeria ivanovii et Listeria innocua, which are the species mostcommonly isolated, and which have enzyme profiles closely resemblingthat of Listeria monocytogenes.

To this effect, this invention concerns a substrate which can be used todirectly identify pathogenic bacteria belonging to the genus Listeria,characterized in that it detects a Listeria monocytogenes-specificesterase activity, an esterase activity which is distinct from PI-PLCactivity.

Thus although PI-PLC is an estérase, Listeria ivanovii is esterasenegative despite being PI-PLC positive; therefore it could be confusedwith Listeria monocytogenes. In contrast to the background art andmethods based on the detection of PI-PLC activity which do notdifferentiate Listeria monocytogenes from other Listeria species, thisinvention makes it possible to detect Listeria monocytogenes in a morespecific way.

According to a preferred embodiment, the esterase activity is a specificenzyme activity, i.e. it cleaves the ester linkage between the markerpart and the target part of the substrate.

According to a preferred embodiment, the linkage cleaved is an esterbond between an alcohol group on the marker part and an organic acidwhich constitutes the target.

According to a preferred embodiment, the marker part consists of achromogenic molecule such as indoxyl which could be constituted by oneof the following constituents:

-   -   5-Bromo-3-indoxyl, or    -   5-Bromo-4-chloro-3-indoxyl, or    -   6-Chloro-3-indoxyl, or    -   5-Bromo-6-chloro-3-indoxyl, or    -   6-Bromo-3-indoxyl.

According to a preferred embodiment, the target part consists of a fattyacid with a carbon chain containing between 2 and 20 carbon atoms,preferably between 4 and 10 carbon atoms.

Preferably, the substrate is either 5-Bromo-4-chloro-3-indolyl butyrate,5-Bromo-4-chloro-3-indolyl octanoate, 5-Bromo-4-chloro-3-indolylnonanoate or 5-Bromo-4-chloro-3-indolyl decanoate.

According to a modified embodiment, the substrate is paired with atleast one other substrate making it possible to detect at least oneother enzyme activity expressed by all or some Listeria species.

In the case in which there are two substrates, the substrate to detectesterase activity other than PI-PLC gives a color which is differentfrom that given by the other enzyme activity which is different from theabove-mentioned esterase activity.

According to a preferred embodiment, the other enzyme activity expressedby all or some Listeria species is a saccharidase, a phosphatase or anaminopeptidase.

Preferably, the marker part of the other substrate is based on:

-   -   5-Bromo-3-indoxyl, or    -   5-Bromo-4-chloro-3-indoxyl, or    -   6-Chloro-3-indoxyl, or    -   5-Bromo-6-chloro-3-indoxyl, or    -   6-Bromo-3-indoxyl.        With respect to -Chloro-3-indoxyl, such a molecule is        particularly well described in Patent U.S. Pat. No. 5,364,767 in        which it is essentially associated with        N-Acetyl-β-D-galactosaminide, with N-Acetyl-β-D-glucosaminide,        with Butyrate, with Octanoate, with the salt of p-toluidine        phosphate, with Sulfate or with β-D-Glucopyranoside. It can also        be based on 5-Bromo-6-chloro-3-indoxyl.

According to a modified embodiment, the other substrate consists of:

-   -   5-Bromo-6-chloro-3-indolyl-β-D-glucoside, or    -   6-Chloro-3-indolyl-β-D-glucoside, or    -   6-Chloro-3-indolyl-β-D-cellobioside, or    -   6-Chloro-3-indolyl-N-acetyl-β-D-glucosaminide, or    -   6-Chloro-3-indolyl-α-D-mannoside, or    -   6-Chloro-3-indolylphosphate.

This invention also concerns a reaction medium which allows the directidentification of Listeria monocytogenes, which exploits one or twosubstrates as defined above.

More precisely, the substrate which detects the esterase activity otherthan PI-PLC is at a concentration of between 20 mg/l and 3 g/l, orpreferably between 50 mgL and 1 g/l, or preferably between 100 and 600mg/l, or about 250 mg/l.

More precisely, the substrate which detects the other activity such as asaccharidase, a phosphatase or an aminopeptidase is at a concentrationof between 10 mg/l and 500 mg/l, preferably between 50 and 300 mg/l, andmore preferably still between 100 and 200 mg/l.

The medium is either liquid or semi-solid (with agar).

According to a modified embodiment, the medium includes a means ofdistinguishing Listeria monocytogenes from Listeria weishimeri andListeria seeligeri, namely:

-   -   the addition of at least one acidified carbohydrate (e.g. Xylose        and/or D-Tagatose) by Listeria welshimeri and Listeria seeligeri        and not by Listeria monocytogenes, or    -   the addition of at least one acidified carbohydrate by Listeria        monocytogenes and possibly Listeria innocua and/or Listeria        ivanovii and/or Listeria grayii.    -   the addition of at least one substrate to detect saccharidase        activity (β-maltosidase) and/or phosphatase and/or        aminopeptidase activity (L-glycine aminopeptidase) specific at        least to Listeria welshimeri and Listeria seeligeri but not        Listeria monocytogenes, or    -   the addition of a natural Phospholipase (PLC) substrate, such as        Phosphatidylinositol (PI) and/or Phosphatidylcholine (PC).

According to a particular embodiment, the medium includes a selectivemeans of differentiating Listeria monocytogenes from at least thefollowing species:

-   -   Staphylococcus aureus,    -   Bacillus thuringiensis,    -   Enterococcus faecalis,    -   Escherichia coli,    -   Pseudomonas aeruginosa, and    -   Candida albicans.

According to a preferred embodiment, the selective means is constitutedby one of the following compounds:

-   -   Lithium chloride    -   Ceftazidime,    -   Amphotericin B,    -   Fosfomycin, and/or    -   Colistin.

This invention furthermore concerns a method for identifying pathogenicbacteria belonging to the species Listeria monocytogenes, comprising thefollowing steps:

-   -   seeding of a culture medium as described above with a specimen        suspected of containing the pathogenic bacteria,    -   incubation of the culture medium seeded with the specimen, and    -   determination of the presence of said pathogenic bacteria by        virtue of the color and intensity typical of the substrate(s).

According to a modified embodiment, said specimen suspected ofcontaining pathogenic bacteria is preliminarily concentrated prior toseeding on the culture medium, as defined above.

According to another modified embodiment of the method, whether or notsaid pathogenic bacteria are present is determined by virtue of thecolor and intensity typical of the substrate(s) after a period of 18 to24 hours of incubation.

Finally, the invention proposes using a substrate consisting of a targetpart as defined above and an inhibitory part which specifically inhibitsListeria monocytogenes when it is released.

This invention therefore essentially concerns the differentiation ofListeria monocytogenes from other species belonging to the genusListeria.

Esterase enzyme activity is a good marker for differentiating thespecies Listeria monocytogenes from other species belonging to the genusListeria, apart from the more specific esterase activity referred to asPI-PLC.

Thus, the background art described above demonstrates the lack ofspecificity of PI-PLC (which is specific for a certain type of lipid)vis-à-vis differentiating the species Listeria monocytogenes andListeria ivanovii, for example. PI-PLC hydrolyzes Phosphatidylinositolderivatives at a point between the Glycerol and the inorganic Phosphate.In the case of a chromogenic substrate, the enzyme cleaves between themarker part and the inorganic Phosphate bound to the Inositol, asillustrated below.

Esterases according to this invention hydrolyze lipids containing one ormore fatty acids with a chain preferably containing between 7 and 10carbon atoms. These esterases hydrolyze ester bonds between an alcoholand an organic fatty acid, as illustrated below.

EXPERIMENT 1 Testing Several Esterase Substrates with the SameChromogenic Marker but Fatty Acids with Different Chain Lengths

The chromogenic esterase substrates tested were:

-   -   5-Bromo-3-indolyl octanoate, hereafter referred to as Blue-C8,    -   5-Bromo-3-indolyl nonanoate, hereafter referred to as Blue-C9,        and    -   5-Bromo-3-indolyl decanoate, hereafter referred to as Blue-C10,

A stock solution of each substrate at a concentration of 40 g/l was madeup in a mixture of 40% Dimethylsulfoxide and 60% PolyoxyethylenesorbitanMonolaurate (Tween 20). An appropriate volume was added to threeColumbia-type media while cooling to give a final substrateconcentration of 250 mg/l. These dishes were divided into three areasand then each area was inoculated with a suspension (density=0.5McFarland) of microorganisms taken from either the Applicant's owncollection or the ATCC. The dishes were incubated for 48 hours at 37° C.The colonies which grew were examined by eye after 24 and 48 hours ofincubation. Both the color and the intensity of the color of thecolonies were recorded. The results are presented in Table 1 below.TABLE 1 Testing several esterase substrates with the same chromogenicmarker but containing fatty acids with different chain lengths with withwith Medium Blue-C8 Blue-C9 Blue-C10 Strain IT C I C I C I Listeriamonocytogenes 24 h gray 1 gray 1 gray 0.5 ATCC 19115 48 h gray 2 gray 2gray 1 Listeria monocytogenes 24 h gray 1 gray 1 gray 0.5 ATCC 19117 48h gray 2 gray 1.5 gray 1 Listeria monocytogenes 24 h gray 1.5 gray 1gray 1 092 48 h gray 2.5 gray 2 gray 2 Listeria innocua 24 h gray 0.3 —— — — 166 48 h gray 1 gray 0.5 gray 0.5 Listeria innocua 24 h gray 0.3 —— — — ATCC 33090 48 h gray 0.5 gray 0.3 gray 0.3 Listeria innocua 24 h —— — — — — 171 48 h gray 0.3 gray 0.3 gray 0.3 Listeria seeligeri 24 h —— — — — — 011 48 h gray 0.5 gray 0.5 gray 0.5 Listeria welshimeri 24 hgray 0.5 gray 0.5 — — 078 48 h gray 1 gray 1 gray 0.5 Listeria grayi 24h — — — — — — ATCC 19120 48 h — — — — — — Listeria ivanovii 24 h — — — —— — 018 48 h — — — — — — Listeria ivanovii 24 h — — — — — — 025 48 h — —— — — —

In Table 1 above, as in the following Tables, C represents the color ofthe colonies after incubation, I represents the intensity of the color,and the symbol “-” corresponds to the absence of color or intensity;finally, IT defines the incubation time. It should be noted that theintensity of the color is scored on an arbitrary scale but one which isconsistent for all the biological specimens and media tested. This scaleis valid for this experiment as well as all those that follow. It can bedefined in the following way:

-   -   0 corresponds to no activity,    -   0.1 corresponds to a trace of color,    -   0.5 corresponds to very pale coloration,    -   1 corresponds to clear but low-intensity coloration,    -   1.5 corresponds to a degree of coloration intermediate between        scores 1 and 2,    -   2 corresponds to clear coloration of moderate intensity,    -   2.5 corresponds to a degree of coloration intermediate between        scores 2 and 3,    -   3 corresponds to intense coloration,    -   3.5 corresponds to a degree of coloration intermediate between        scores 3 and 4,    -   4 corresponds to very intense coloration.

Only strains of L. monocytogenes, L. innocua, L. seeligeri and L.welshimeri gave a gray color: these strains therefore express anesterase activity. It is possible to distinguish—whatever the length ofthe fatty acid chain being tested—a difference of about 1 unit in theintensity given by L. monocytogenes strains (the higher scores) and theintensities given by strains of L. innocua, L. welshimeri and L.seeligeri.

However, in the following examples, only the results obtained withsubstrates based on the octanoate and the nonanoate will be given.

Moreover, it can be seen that only strains of Listeria monocytogenesgive any color after 24 hours. It is therefore possible to differentiateListeria monocytogenes from other Listeria species on the basis ofincubation temperature.

EXPERIMENT 2 Testing Several Esterase Substrates with DifferentIndoxyl-based Chromogenic Markers and with Octanoic Acid as the FattyAcid

The chromogenic esterase substrates tested were:

-   -   5-Bromo-3-indolyl octanoate, hereafter referred to as Blue-C8,    -   5-Bromo-4-chloro-3-indolyl octanoate, hereafter referred to as        X-C8,    -   6-Chloro-3-indolyl octanoate, hereafter referred to as Pink-C8;        and    -   5-Bromo-6-chloro-3-indolyl octanoate, hereafter referred to as        Magenta-C8,

A stock solution of each substrate at a concentration of 40 g/l was madeup in a mixture of 40% Dimethylsulfoxide and 60% Tween 20. Anappropriate volume was added to four Columbia-type media while coolingto give a final substrate concentration of 250 mg/l. These dishes weredivided into three areas and then each area was inoculated with asuspension (density=0.5 McFarland) of microorganisms taken from eitherthe Applicant's own collection or the ATCC. The dishes were incubatedfor 48 hours at 37° C. The colonies which grew were examined by eyeafter 24 and 48 hours of incubation. Both the color and the intensity ofthe color of the colonies were recorded. The results are presented inTable 2 below. TABLE 2 Results obtained with several esterase substrateswith different Indoxyl-based chromogenic markers and with octanoic acidas the fatty acid with with Blue- with Pink- Magenta- C8 with X-C8 C8 C8Strain Medium IT C I C I C I C I Listeria 24 h gray 1 turquoise 2.5 pink0.5 purple 2 monocytogenes ATCC 19115 48 h gray 2 turquoise 3 pink 1purple 3 Listeria 24 h gray 1 turquoise 2 pink 0.5 purple 1.5monocytogenes ATCC 19117 48 h gray 2 turquoise 3 pink 1 purple 3.5Listeria 24 h gray 1.5 turquoise 3 pink 1.5 purple 3 monocytogenes 09248 h gray 2.5 turquoise 3.5 pink 2 purple 3 Listeria innocua 24 h gray0.3 turquoise 0.3 — — purple 2 166 48 h gray 1 turquoise 0.5 pink 0.3purple 3 Listeria innocua 24 h gray 0.3 turquoise 0.3 — — purple 1 ATCC33090 48 h gray 0.5 turquoise 0.5 pink 0.5 purple 2 Listeria innocua 24h — — — — — purple 0.5 171 48 h gray 0.3 turquoise 0.3 pink 0.3 purple 3Listeria ivanovii 24 h — — — — — — — — 018 48 h — — — — — — — — Listeriaivanovii 24 h — — — — — — — — 025 48 h — — — — — — — —

Whatever substrate was used, a difference in intensity was observedbetween strains of Listeria monocytogenes and strains of Listeriainnocua. However, the difference was more significant for the markers5-Bromo-4-chloro-3-indoxyl.

Beter contrast and stronger intensity was obtained with the marker thatgave a turquoise color, namely 5Bromo-4-chloro-3-indoxyl.

EXPERIMENT 3 Detecting an Esterase Activity in Liquid Medium

The chromogenic esterase substrates tested were:

-   -   5-Bromo-4-chloro-3-indolyl acetate, hereafter referred to as        X-C2,    -   3-Indolyl acetate, hereafter referred to as Y-C2,    -   5-Bromo-4-chloro-3-indolyl butyrate, hereafter referred to as        X-C4,    -   5-Bromo-4-chloro-3-indolyl octanoate, hereafter referred to as        X-C8,    -   5-Bromo-6-chloro-3-indolyl octanoate, hereafter referred to as        M-C8, and    -   6-Chloro-3-indolyl octanoate, hereafter referred to as R-C8.

These substrates were lyophilized in the wells of API-type strips(Registered Trademark). Before use, the substrates contained in thewells were resuspended in an agar-free Columbia-type inoculation medium.These wells were then inoculated with a suspension (density=2 McFarland)of microorganisms taken from either the Applicant's own collection orthe ATCC. The strips were incubated for 8 hours at 37° C. The colonieswhich grew were examined by eye after 4, 6 and 8 hours of incubation.

The intensity of the color of the colonies was recorded. The results arepresented in Table 3 below. TABLE 3 Results obtained with severalesterase substrates with different Indoxyl-based chromogenic markers andwith fatty acids containing chains of different lengths Medium X-C2 Y-C2X-C4 X-C8 M-C8 R-C8 Strain IT I I I I I I Listeria 4 h 3 3 1 — — —monocytogenes 6 h 4 4 2 1 — — ATCC 19115 8 h 4 4 3 1.5 0.5 — Listeria 4h 3 4 2 — — — monocytogenes 6 h 4 4 2 1 — — 092 8 h 4 4 3 1.5 0.5 —Listeria innocua 171 4 h 4 3 0.5 — — — 6 h 4 3 1 — — — 8 h 4 4 2 — — —Listeria innocua 166 4 h 3 3 0.5 — — — 6 h 4 3 1 — — — 8 h 4 4 2 — — —Listeria welshimeri 4 h 3 3 0.5 — — — 023 6 h 4 4 1 — — — 8 h 4 4 2 — —— Listeria seeligeri 4 h 3 3 0.5 — — — 011 6 h 3 3 1 0.5 — — 8 h 4 4 20.5 — — Listeria ivanovii 018 4 h 3 3 0.5 — — — 6 h 3 4 1 — — — 8 h 4 42 — — — Listeria grayi 4 h 2 3 0.5 — — — ATCC 6 h 2 4 1 — — — 19120 8 h3 4 2 — — —

In liquid medium, substrates with a chain of under 4 carbon atoms do notdifferentiate between different Listeria species. In contrast, in theoperating conditions tested, the substrates X-C4 and X-C8 gave acontrast in color between L. monocytogenes and other species belongingto the same genus. In practice, only L. welshimeri and L. seeligeri alsogave any color, but the color that they gave was of very low intensity.It is possible to imagine setting a threshold for a positive responseat 1. Test strains which gave a reading of below 1 would be consideredas negative; strains giving a reading of over 1 would be considered aspositive. Thus, in the present case, it would mean that it is a strainof L. monocytogenes.

EXPERIMENT 4 Simultaneous Detection of an Esterase Activity and aSaccharidase Activity in Semi-solid Medium

Various pairs of a chromogenic esterase substrate with a chromogenicsaccharidase substrate were tested, as follows:

-   5-Bromo-3-indolyl octanoate and 6-Chloro-3-indolyl-β-D-glucoside,    which corresponds to Pair 1,-   5-Bromo-3-indolyl octanoate and 6-Chloro-3-indolyl-β-D-cellobioside,    which corresponds to Pair 2,-   5-Bromo-3-indolyl octanoate and    6-Chloro-3-indolyl-N-acétyl-β-D-glucosaminide, which corresponds to    Pair 3,-   5-Bromo-3-indolyl octanoate and 6-Chloro-3-indolyl-α-D-mannoside,    which corresponds to Pair 4,-   5-Bromo-4-chloro-3-indolyl octanoate and    6-Chloro-3-indolyl-β-D-glucoside, which corresponds to Pair 5,-   5-Bromo-4-chloro-3-indolyl octanoate and    6-Chloro-3-indolyl-β-D-cellobioside, which corresponds to Pair 6,-   5-Bromo-4-chloro-3-indolyl octanoate and    6-Chloro-3-indolyl-N-acétyl-β-D-glucosaminide, which corresponds to    Pair 7, and-   5-Bromo-4-chloro-3-indolyl octanoate and    6-Chloro-3-indolyl-α-D-mannoside, which corresponds to Pair 8.

A stock solution of each esterase substrate at a concentration of 40 g/lwas made up in a mixture of 40% Dimethylsulfoxide and 60% Tween 20. Anda stock solution of each saccharidase substrate was made up inDimethylsulfoxide. An appropriate volume of the stock solution of theesterase substrate corresponding to each of the pairs described abovewas added to one of eight Columbia-type media while cooling to obtain afinal substrate concentration of 250 mg/l. In parallel, an appropriatevolume of the stock solution of the saccharidase substrate correspondingto each of the pairs described above was added to each of the eightmedia in order to obtain a final concentration of between 100 and 200mg/l (depending on the substrate). These dishes were divided into threeareas and then each area was inoculated with a suspension (density=0.5McFarland) of microorganisms taken from either the Applicant's owncollection or the ATCC. The dishes were incubated for 48 hours at 37° C.The colonies which grew were examined by eye after 24 and 48 hours ofincubation.

Both the color and the intensity of the color of the colonies wererecorded. The results are presented in Table 4 below. TABLE 4 Resultsobtained using different pairs of chromogenic substrates tosimultaneously detect an esterase activity and a saccharidase activityin a semi-solid medium Pair 1 Pair 2 Pair 3 Pair 4 Strain Medium IT C IC I C I C I Listeria 24 h gray 2 purple- 2 purple 3 purple 3monocytogenes gray ATCC 19115 48 h purple- 3 purple- 3 purple 3.5 purple3.5 gray gray Listeria 24 h purple- 2 purple- 2 purple 3 purple 2monocytogenes gray gray ATCC 19117 48 h purple- 3 purple- 3 purple- 3.5purple 3.5 gray gray gray Listeria 24 h purple- 3 purple- 2 purple 3purple 3 monocytogenes gray gray 092 48 h purple- 3.5 purple- 3 purple-3.5 purple 3.5 gray gray gray Listeria innocua 24 h mauve 1.5 pink-gray1.5 mauve 2 mauve 2 166 48 h mauve 3 pink-gray 2 purple 3 purple 3Listeria innocua 24 h mauve 1 pink-gray 1 mauve 2 mauve 2 ATCC 33090 48h mauve 3 pink-gray 2 purple 3 purple 3 Listeria innocua 24 h mauve 1pink-gray 1 mauve 1.5 mauve 1.5 171 48 h mauve 3 pink-gray 2 purple 3purple 3 Listeria ivanovii 24 h pink 1 pink 1 pink 1 pink 1 018 48 hpink 2 pink 2 pink 2 pink 2 Listeria ivanovii 24 h pink 1 pink 1 pink 1pink 1 025 48 h pink 2 pink 2 pink 2 pink 2 Medium Pair 5 Pair 6 Pair 7Pair 8 Strain TI C I C I C I C I Listeria 24 h blue 3 blue 3 blue 3 blue3 monocytogenes 48 h blue- 3.5 blue-gray 3.5 blue- 3.5 blue- 3.5 ATCC19115 gray gray gray Listeria 24 h blue 3 blue 3 blue 3 blue 3monocytogenes 48 h blue- 3.5 blue-gray 3.5 blue- 3 blue- 3.5 ATCC 19117gray gray gray Listeria 24 h blue 3 blue 3 blue 3 blue 3 monocytogenes48 h blue- 3.5 blue-gray 3.5 blue- 3.5 blue- 4 092 gray gray grayListeria innocua 24 h purple 1 blue- 1 mauve 1 purple 2 166 purple 48 hpurple 2 blue- 2 mauve 2 purple 3 purple Listeria innocua 24 h purple 1blue- 2 mauve 1 purple 2 ATCC 33090 purple 48 h purple 3 blue- 3 mauve 3purple 3 purple Listeria innocua 24 h purple 1 blue- 2 mauve 1 purple 2171 purple 48 h purple 3 blue- 2 mauve 3 purple 3 purple Listeriaivanovii 24 h pink 1 pink 1 pink 1 pink 1 018 48 h pink 2 pink 2 pink 2pink 2 Listeria ivanovii 24 h pink 1 pink 1 pink 1 pink 1 025 48 h pink2 pink 2 pink 2 pink 2

Whatever pair of substrates was tested, a color difference was observedbetween strains of Listeria monocytogenes and strains of Listeriainnocua and Listeria ivanovii.

The best contrast was obtained with 5-Bromo-4-chloro-3-indolyl nonanoatetogether with a saccharidase substrate based on 6-Chloro-3-indolyl,whatever the target activity. Therefore, in principle any of the variousesterase substrates can be paired with a saccharidase, be it anα-polysaccharidases or a β-polysaccharidase.

However, it can be seen that the best results were obtained using6-Chloro-3-indolyl-β-D-glucoside,6-Chloro-3-indolyl-N-acetyl-β-D-glucosaminide and6-Chloro-3-indolyl-α-D-mannoside.

EXPERIMENT 5 Simultaneous Detection of an Esterase Activity and aPhosphatase Activity in Semi-solid Medium

Volumes of two stock solutions were successively added into a semi-solidColumbia-type medium while cooling: 5-Bromo-4-chloro-3-indolyl octanoate(X-C8) (final concentration in the medium: 250 mg/l) and6-Chloro-3-indolyl phosphate (Pink P) (final concentration in themedium: 750 mg/l). The stock 5-Bromo-4-chloro-3-indolyl octanoatesolution was prepared as in the previous examples, and that of6-Chloro-3-indolyl phosphate was made up at a concentration of 50 g/l inDimethylsulfoxide.

These dishes were divided into three areas and then each area wasinoculated with a suspension (density=0.5 McFarland) of microorganismstaken from either the Applicant's own collection or the ATCC. The disheswere incubated for 48 hours at 37° C. The colonies which grew wereexamined by eye after 24 and 48 hours of incubation.

Both the color and the intensity of the color of the colonies wererecorded. The results are presented in Table 5 below. TABLE 5simultaneous detection of an esterase activity and a phosphataseactivity Medium X-C8 and Pink P Strain IT C I Listeria 24 h blue-gray 2monocytogenes 48 h blue-gray 3 ATCC 19115 Listeria 24 h blue 2monocytogenes 48 h blue-gray 3 092 Listeria innocua 24 h pink-gray 0.5166 48 h pink-gray 2 Listeria innocua 24 h pink 0.5 171 48 h pink 1.5Listeria ivanovii 24 h pink-gray 3 022 48 h pink-gray 3 Listeriawelshimeri 24 h blue-gray 3 081 48 h blue-gray 3 Listeria seeligeri 24 hblue-gray 0.5 011 48 h blue-gray 2 Listeria grayi 24 h gray 0.5 ATCC19120 48 h gray 1

Simultaneous testing of both these activities make it possible todifferentiate between two groups of Listeria, on the one hand L.monocytogenes, L. seeligeri and L. welshimeri, and on the other hand L.grayi, L. innocua and L. ivanovii. This detection system can be improvedby adjusting the composition of the medium (as will be explained inExperiment 6) so that it becomes possible to differentiate L.monocytogenes from all other species belonging to the genus Listeria.

EXPERIMENT 6 Differentiation of Listeria monocytogenes from Listeriawelshimeri and Listeria seeligeri in a Semi-solid Medium Containing anEsterase Substrate and a Saccharidase Substrate

The following pair of chromogenic esterase substrate with chromogenicsaccharidase substrate was tested: 5-Bromo-4-chloro-3-indolyl octanoateand 6-Chloro-3-indolyl-N-acetyl-β-D-glucosaminide

Media and stock substrate solutions were prepared as in the previousexamples. This basic medium was divided into four portions into whichwere added respectively:

-   -   40 g/l Xylose corresponding to Medium 1,    -   40 g/l Tagatose corresponding to Medium 2,    -   a mixture of 45 g/l carbohydrates with 30 g/l Xylose and 15 g/l        Tagatose corresponding to Medium 3,    -   a mixture of 65 g/l carbohydrates with 35 g/l Xylose and 30 g/l        Tagatose corresponding to Medium 4,

These dishes were divided into three areas and then each area wasinoculated with a suspension (density=0.5 McFarland) of microorganismstaken from the Applicant's collection. The dishes were incubated for 48hours at 37° C. The colonies which grew were examined by eye after 24and 48 hours of incubation.

Both the color and the intensity of the color of the colonies wererecorded. The results are presented in Table 6 below. TABLE 6Enhancement of the specificity for L. monocytogenes vis-à-vis L.welshimeri and L. seeligeri by the addition of a very high concentrationof carbohydrate Medium 1 Medium 2 Medium 3 Medium 4 Strain Medium IT C IC I C I C I Listeria 24 h blue 2 blue- 1 blue- 1 blue- 0.5 monocytogenesgray gray gray 079 48 h blue- 3 blue- 3 blue- 3 blue- 3 gray gray graygray Listeria 24 h blue 2 blue- 1 blue- 1 blue- 0.5 monocytogenes graygray gray 081 48 h blue- 3 blue- 3 blue- 3 blue- 3 gray gray gray grayListeria innocua 24 h mauve 2 pink 1 pink 1 mauve 0.5 29 48 h mauve 2mauve 3 mauve 3 mauve 3 Listeria seeligeri 24 h turquoise 2 turquoise0.5 turquoise 0.5 turquoise 0.5 012 48 h turquoise 3 blue- 3 turquoise 3turquoise 2 gray Listeria welshimeri 24 h turquoise 2 turquoise 0.5turquoise 0.5 turquoise 0.5 023 48 h blue- 3 blue- 3 turquoise 3turquoise 3 gray gray Listeria ivanovii 24 h pink 1 pink 1 pink 1 pink 1025 48 h pink 2 pink 2 pink 2 pink 2 Listeria grayi 24 h pink 3 pink 3pink 3 pink 3 078 48 h pink 3 pink 3 pink 3 pink 3

Adding a very high concentration of carbohydrate (over 40 g/l) makes itpossible to make a distinction between L. monocytogenes and all otherListeria species, and this purely on the basis of color. Nevertheless,it has been shown that it is possible to make a distinction betweenspecies using lower concentrations (down to 10 g/l) as is detailed inPatent FR-B-2.708.285. However here, unlike in this above-mentionedPatent, the color change is due to the inhibition of an enzyme, not dueto a derived color different from the basic color of the marker.

EXPERIMENT 7 Addition of Selective Agents to Inhibit Gram-positiveBacteria Other Than Listeria, and to Inhibit Gram-negative Bacteria andYeast

The basic Columbia-type medium contains:

-   -   5-Bromo-4-chloro-3-indolyl octanoate (250 mg/l) prepared from a        stock solution in a mixture of 40% Dimethylsulfoxide and 60%        Polyoxyethylenesorbitan Monolaurate (Tween 20),    -   5-Bromo-6-chloro-3-indolyl-?-N-acétylglucosaminide (150 mg/l)        prepared from a stock solution in DMSO,    -   Xylose (10 g/l) and Tagatose (5 g/l), and    -   a selective medium defined as follows: Lithium chloride (5 g/l),        Ceftazidime (20 mg/l), Amphotericin B (4 mg/l), Fosfomycin (10        mg/l) and Colistin (5 mg/l).

These dishes were divided into three areas and then each area wasinoculated with a suspension (density=0.5 McFarland) of microorganismstaken from the Applicant's collection. The dishes were incubated for 48hours at 37° C. The colonies which grew were examined by eye after 24and 48 hours of incubation. Both the color and the intensity of thecolor of the colonies were recorded. The results are presented in Table7 below. TABLE 7 Results obtained with media either containing or notcontaining selective agents no selective with selective agent agentsStrain Medium IT C I C I Listeria 24 h blue 1 blue 1 monocytogenes 48 hblue 3 blue 3 028 Listeria 24 h blue 2 blue 2 monocytogenes 48 h blue3.5 blue 3 023 Listeria innocua 24 h mauve 1.5 mauve 1 036 48 h mauve 3mauve 2 Listeria ivanovii 24 h pink 1 — — 018 48 h pink 2 mauve 1Listeria welshimeri 24 h turquoise 1 turquoise 1 081 48 h turquoise 3turquoise 2 Staphylococcus aureus 24 h green 2 — — 029 48 h green 3.5 —— Bacillus thuringiensis 24 h turquoise 0.5 072 48 h turquoise 1Enterococcus faecalis 24 h turquoise 0.5 — — 117 48 h turquoise 1 — —Escherichia coli 24 h colorless — — — 006 48 h colorless — — —Pseudomonas 24 h green 0.5 — — aeruginosa 48 h green 2 — — 003 Candidaalbicans 24 h turquoise 0.5 — — 077 48 h turquoise 1 — —

In Table 7 above, C represents the color of colonies after incubation, Irepresents the intensity of this color, the symbol “-” signals theabsence of any growth, and finally IT corresponds to incubation time. Itshould be noted that the intensity of the color is scored on anarbitrary scale but one which is consistent for all the biologicalspecimens and media tested.

It can be seen that, in the presence of selective agents, all theinterfering bacteria and yeast present (i.e. any microorganisms whichare not Listeria) fail to grow, i.e. are inhibited. The presence ofselective agents does not affect the color of the Listeria strainstested.

This experiment also shows that, even without any selective agents, itis possible to differentiate between Listeria monocytogenes on the onehand and interfering bacteria and yeast on the other hand, which is nottrue with the substrates, culture media and reagents described in thebackground art.

1-22. (canceled)
 23. A method for identifying pathogenic bacteria of thespecies Listeria monocytogenes, comprising the following steps: seedingof a culture medium with a specimen suspected of containing thepathogenic bacteria, incubation of the culture medium seeded with thespecimen, and determination of the presence of said pathogenic bacteriaby virtue of the color and intensity typical of the substrate(s),wherein said culture medium comprises a reaction medium for the directidentification of pathogenic bacteria belonging to the species Listeriamonocytogenes, which exploits at least one substrate which is cleaved byan esterase activity other than Phosphatidyl Inositol-specificPhospholipase C (PI-PLC), an esterase activity which is specific forListeria monocytogenes and not specific for other members of the genusListeria.
 24. The method of claim 23, wherein said specimen suspected ofcontaining the pathogenic bacteria is preliminarily concentrated beforethe seeding of said culture medium.
 25. The method of claim 23, whereinwhether or not said pathogenic bacteria are present is determined byvirtue of the color and intensity typical of the substrate(s) after aperiod of 18 to 24 hours of incubation.